Mitochondrial diseases are devastating disorders for which there is no cure and no proven treatment. About 1 in 2000 individuals are at risk of developing a mitochondrial disease sometime in their lifetime. Half of those affected are children who show symptoms before age 5 and approximately 80% of these will die before age 20. The human suffering imposed by mitochondrial and metabolic diseases is enormous, yet much work is needed to understand the genetic and environmental causes of these diseases. Mitochondrial genetic diseases are characterized by alterations in the mitochondrial genome, as point mutations, deletions, rearrangements, or depletion of the mitochondrial DNA (mtDNA). The mutation rate of the mitochondrial genome is 10-20 times greater than of nuclear DNA, and mtDNA is more prone to oxidative damage than is nuclear DNA. Mutations in human mtDNA cause premature aging, severe neuromuscular pathologies and maternally inherited metabolic diseases, and influence apoptosis. The primary goal of this project is to understand the contribution of the replication apparatus in the production and prevention of mutations in mtDNA. Since the genetic stability of mitochondrial DNA depends on the accuracy of DNA polymerase gamma (pol gamma), we have focused this project on understanding the role of the human pol gamma in mtDNA mutagenesis. Human mitochondrial DNA is replicated by the two-subunit gamma, composed of a 140 kDa subunit containing catalytic activity and a 55 kDa accessory subunit. The catalytic subunit contains DNA polymerase activity, 3'-5'exonuclease proofreading activity, and 5'dRP lyase activity required for base excision repair. As the only DNA polymerase in animal cell mitochondria, pol gamma participates in DNA replication and DNA repair. The 140 kDa catalytic subunit for pol gamma is encoded by the nuclear POLG gene. To date nearly 200 pathogenic mutations in POLG that cause a wide spectrum of disease including Progressive external ophthalmoplegia (PEO), parkinsonism, premature menopause, Alpers syndrome, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) or sensory ataxic neuropathy, dysarthria, and ophthalmoparesis (SANDO). Presently, there are over 150 pathogenic disease mutations in the POLG gene that cause PEO, ataxia-neuropathy and Alpers syndrome. The c.1550 g&#8594;t mutation in the POLG gene causing the G517V substitution has been reported by many groups to be associated with a variety of mitochondrial diseases, including autosomal dominant and recessive forms of ataxia neuropathy, myopathy and microcephaly, progressive external ophthalmoplegia, diabetes, strokes, hypotonia, and epilepsy. However, the variable disease presentation and age of onset raises suspicion of its pathogenicity. Because of the varied reported associated symptoms and request from physicians to address the consequence of this mutation, we have carried out the biochemical analysis of the purified recombinant human DNA polymerase &#947;protein harboring the G517V substitution. These analyses revealed that the G517V mutant enzyme retained 80-90% of wild-type DNA polymerase activity, in addition to its functional interaction with the p55 accessory subunit. DNA binding by the mutant was also only slightly lower than the wild-type enzyme. Our data suggest that the G517V mutation by itself in pol &#947;most likely does not have a role in mitochondrial disorders. Defects in mitochondrial DNA (mtDNA) maintenance comprise an expanding repertoire of polymorphic diseases caused, in part, by mutations in the genes encoding the p140 mtDNA polymerase (POLG), its p55 accessory subunit (POLG2) or the mtDNA helicase (C10orf2). In an exploration of nuclear genes for mtDNA maintenance linked to mitochondrial disease, 8 heterozygous mutations (6 novel) in POLG2 were identified in 1 control and 8 patients with POLG-related mitochondrial disease that lacked POLG mutations. Of these eight mutations we biochemically characterized 7 variants (c.307G>A (G103S);c.457C>G (L153V);c.614C>G (P205R);c.1105A>G (R369G);c.1158T>G (D386E);c.1268C>A (S423Y);c.1423_1424delTT (L475DfsX2)) that were previously uncharacterized along with the wild-type protein and the G451E pathogenic variant. These seven mutations encode amino acid substitutions that map throughout the protein, including the p55 dimer interface and the C-terminal domain that interacts with the catalytic subunit. Recombinant proteins harboring these alterations were assessed for stimulation of processive DNA synthesis, binding to the p140 catalytic subunit, binding to dsDNA, and self-dimerization. Whereas the G103S, L153V, D386E and S423Y proteins displayed wild-type behavior, the P205R and R369G p55 variants had reduced stimulation of processivity and decreased affinity for the catalytic subunit. Additionally, the L475DfsX2 variant, which possesses a C-terminal truncation, was unable to bind the p140 catalytic subunit, unable to bind dsDNA and formed aberrant oligomeric complexes. Our biochemical analysis helps to explain the pathogenesis of POLG2 mutations in mitochondrial disease and emphasizes the need to quantitatively characterize the biochemical consequences of newly discovered mutations before classifying them as pathogenic. The presence of RNA in mitochondrial DNA has been known for several decades but the origin has been a topic of controversy. The existence of RNA in the mitochondrial genome provides opportunities for genomic instability, specifically mediating breaks in DNA, which can lead to deletions. Deletions in mitochondrial DNA are associated with several mitochondrial diseases as well as aging. During DNA synthesis, DNA polymerases must select against ribonucleotides, present at much higher levels compared to deoxyribonucleotides. Most DNA polymerases are equipped to exclude ribonucleotides from its active site through a bulky side chain residue that can sterically block the 2-hydroxyl group of the ribose ring. However, many nuclear replicative and repair DNA polymerases incorporate ribonucleotides into DNA, suggesting that the exclusion mechanism is not perfect. In this study, we show that the human mitochondrial DNA polymerase &#947;discriminates ribonucleotides efficiently but differentially based on the base identity. While, UTP is discriminated by 77,000-fold compared to dTTP, the discrimination drops to 1,100-fold for GTP versus dGTP. In addition, the efficiency of the enzyme reduced 3-14-fold depending on the identity of the incoming nucleotide when it extends from a primer containing a 3-terminal ribonucleotide. DNA polymerase &#947;is also proficient in performing single nucleotide reverse transcription reactions from both DNA and RNA primer terminus, although, its bypass efficiency is significantly diminished with increasing stretches of ribonucleotides in template DNA. Furthermore, we show that the E895A mutant enzyme is compromised in its ability to discriminate ribonucleotides mainly due to its defects in deoxyribonucleoside triphosphate binding and is also a poor reverse transcriptase.